Biology subjects

Kharadi, R. R.

Publications and source records attributed to Kharadi, R. R..

3 recordsLinked to original sources

bioRxiv2024

pGpG-signaling regulates virulence and global transcriptomic targets in Erwinia amylovora

Cyclic-di-GMP (c-di-GMP) is a critical bacterial second messenger that enables the physiological phase transition in Erwinia amylovora, the phytopathogenic bacterium that causes fire blight disease. C-di-GMP generation is dependent on diguanylate cyclase enzymes while the degradation of c-di-GMP can occur through the action of phosphodiesterase (PDE) enzymes that contain an active EAL and/or a HD-GYP domain. The HD-GYP-type PDEs, which are absent in E. amylovora, can directly degrade c-di-GMP into two GMP molecules. PDEs that contain an active EAL domain, as found in all active PDEs in E. amylovora, degrade c-di-GMP into pGpG. The signaling function of pGpG is not fully understood in bacterial systems. A transcriptomic approach revealed that elevated levels of pGpG in E. amylovora impacted several genes involved in metabolic and regulatory functions including several type III secretion and extracellular appendage related genes. The heterologous overexpression of an EAL or HD-GYP-type PDE in different background E. amylovora strains with varying c-di-GMP levels revealed that in contrast to the generation of pGpG, the direct breakdown of c-di-GMP into GMP by the HD-GYP-type PDE led to an elevation in amylovoran production and biofilm formation despite a decrease in c-di-GMP levels. The breakdown of c-di-GMP into pGpG (as opposed to GTP) also led to a decrease in virulence in apple shoots. The expression of hrpS was significantly increased in response to the breakdown of c-di-GMP into pGpG. Further, our model suggests that a balance in the intracellular ratio of pGpG and c-di-GMP is essential for biofilm regulation in E. amylovora. Importancec-di-GMP is the keystone molecule for regulating the transition from motility to biofilm formation in most bacteria. Interestingly, there are two distinct enzymatic phosphodiesterase (PDE) domains, termed EAL and HD-GYP, that degrade c-di-GMP. EAL domains cleave one bond of the cyclic ring to generate pGpG while HD-GYP enzymes cleave c-di-GMP to two GMP molecules. A central question regarding c-di-GMP signaling is has whether or not pGpG itself functions as a signaling molecule. Here we demonstrate in the plant pathogen Erwinia amylovora that pGpG specifically regulates genes and contributes to biofilm formation and disease progression.

microbiology↗

bioRxiv2021

The cyclic di-GMP network is a global regulator of phase-transition and attachment-dependent host colonization in Erwinia amylovora

Cyclic-di-GMP (c-di-GMP) is an essential bacterial second messenger that regulates the transition to biofilm formation in the phytopathogen Erwinia amylovora. The c-di-GMP system in E. amylovora is comprised of 12 diguanylate cyclase/Edc (dimerize cyclic-di-GMP) and phosphodiesterase/Pde (hydrolyze cyclic-di-GMP) proteins that are characterized by the presence of GGDEF and/or EAL motifs in their domain architecture. In order to study the global regulatory effect (without the inclusion of systemic regulatory impedance) of the c-di-GMP system in E. amylovora, we eliminated all 12 edc and pde genes in E. amylovora Ea1189{Delta}12. Comparisons between the representative transcriptomic profiles of Ea1189{Delta}12 and the combinatorial edc gene knockout mutant (Ea1189{Delta}5) revealed marked overall distinctions in expression levels for targets in a wide range of regulatory categories, including metabolic pathways involved in the utilization of methionine, isoleucine, histidine, etc. as well as critical signal transduction pathways including the Rcs phosphorelay and PhoPQ system. A complete loss of the cyclic-di-GMP signaling components resulted in the inability of Ea1189{Delta}12 cells to attach to and form biofilms in vitro and within the xylem vasculature in apple shoots. Using a flow-based in vitro biofilm system, we found that initial surface sensing was primarily dependent on the flagellar filament (FliC), following which the type IV pilus (HofC) was required to anchor cells to the surface to initialize biofilm development. A transcriptomic analysis of WT E. amylovora Ea1189 and Ea1189{Delta}12 cells in various stages of biofilm development revealed that cyclic-di-GMP based regulation had widespread effects on purine and pyrimidine biosynthesis pathways, amylovoran biosynthesis genes and the EnvZ/OmpR signal transduction system. Additionally, complementing individual eliminated genes back into Ea1189{Delta}12, and the collective evaluation of several virulence factors, enabled the correlative clustering of the functional effect rendered by each Edc and Pde enzyme in the system. SignificanceCyclic-di-GMP dependent regulation, in the context of biofilm formation, has been studied in several bacterial systems. However, the comprehensiveness of the studies exploring the role of individual genetic components related to cyclic-di-GMP is affected by the often large number of diguanylate cyclase and phosphodiesterase enzymes present within individual bacterial systems. To explore the evolutionary dependencies related to cyclic-di-GMP in E. amylovora, we used a collective elimination approach, whereby all of the enzymes involved in cyclic-di-GMP metabolism were eliminated from the system. This approach enabled us to highlight the critical importance of cyclic-di-GMP in plant xylem colonization due to its effect on surface attachment. Additionally, we highlight the global transcriptomic effect of cyclic-di-GMP dependent signaling at various stages of biofilm development. Our approach is aimed at exploring the regulatory role of individual cyclic-di-GMP related enzymes in a background that is free from any redundancy-based feedback.

microbiology↗

bioRxiv2019

Cell-length heterogeneity: a population-level solution to growth/virulence trade-offs in the plant pathogen Dickeya dadantii

Necrotrophic plant pathogens acquire nutrients from dead plant cells, which requires the disintegration of the plant cell wall and tissue structures by the pathogen. Infected plants lose tissue integrity and functional immunity as a result, exposing the nutrient rich, decayed tissues to the environment. One challenge for the necrotrophs to successfully cause secondary infection (infection spread from an initially infected plant to the nearby uninfected plants) is to effectively utilize nutrients released from hosts towards building up a large population before other saprophytes come. In this study, we observed that the necrotrophic pathogen Dickeya dadantii exhibited heterogeneity in bacterial cell length in an isogenic population during infection of potato tuber. While some cells were regular rod-shape (<10m), the rest elongated into filamentous cells (>10m). Short cells tended to occur at the interface of healthy and diseased tissues, during the early stage of infection when active attacking and killing is occurring, while filamentous cells tended to form when large amount of nutrients were released at a later stage of infection. Short cells expressed all necessary virulence factors and motility, whereas filamentous cells did not engage in virulence, were non-mobile and more sensitive to environmental stress. However, compared to the short cells, the filamentous cells displayed elevated metabolism and faster growth, which may benefit the pathogens to build up a large population necessary for the secondary infection. The segregation of the two subpopulations was dependent on differential expression of the alarmone guanosine tetraphosphate (ppGpp). When exposed to fresh tuber tissues or freestanding water, filamentous cells quickly transformed to short virulent cells. The pathogen adaptation of cell length heterogeneity identified in this study presents a model for how some necrotrophs balance virulence and vegetative growth to maximize fitness during infection.

microbiology↗